REVIEWS
THE LOCAL DIFFERENTIATION OF
MYELINATED AXONS AT NODES
OF RANVIER
Sebastian Poliak and Elior Peles
Efficient and rapid propagation of action potentials in myelinated axons depends on the
molecular specialization of the nodes of Ranvier. The nodal region is organized into several
distinct domains, each of which contains a unique set of ion channels, cell-adhesion molecules
and cytoplasmic adaptor proteins. Voltage-gated Na+ channels — which are concentrated at the
nodes — are separated from K+ channels — which are clustered at the juxtaparanodal region —
by a specialized axoglial contact that is formed between the axon and the myelinating cell at the
paranodes. This local differentiation of myelinated axons is tightly regulated by oligodendrocytes
and myelinating Schwann cells, and is achieved through complex mechanisms that are used by
another specialized cell–cell contact — the synapse.
Department of Molecular
Cell Biology, The Weizmann
Institute of Science, Rehovot
76100, Israel.
Correspondence to E. P.
e-mail: peles@weizmann.ac.il
doi:10.1038/nrn1253
968
The evolutionary need for the rapid and efficient
conduction of action potentials in vertebrate neurons
has resulted in the development of the myelin sheath.
Myelin, a multilamellar membrane that is formed by
oligodendrocytes in the central nervous system (CNS)
and by Schwann cells in the peripheral nervous system
(PNS), enwraps the axon in segments that are separated
by the nodes of Ranvier (FIG. 1a). The myelin sheath
reduces current flow across the axonal membrane by
reducing its capacitance and increasing its transverse
resistance, thereby allowing the fast, saltatory movement
of nerve impulses from node to node1. As a consequence,
a large number of axons with high conduction velocities
could be placed in a limited space, a feature that permitted the development of more complex nervous systems.
In addition, saltatory conduction eliminates the need
for regenerating the action potential at every point of
axonal membrane, therefore reducing the metabolic
requirements for neuronal activity.
Organization and function of the nodal environs
The coordinated differentiation of the axon and its
myelinating cell requires a close communication between
neurons and glia from early stages of development.
| DECEMBER 2003 | VOLUME 4
Signals provided by the axon regulate the proliferation, survival and differentiation of oligodendrocytes
and Schwann cells2,3, and participate in determining
myelin thickness4. Reciprocal glial signals affect the
axonal cytoskeleton and transport5, and are required
for axonal survival6,7. As a result of this reciprocal
communication, myelinated fibres acquire structural
features that allow them to maximize their conduction
velocities. One such feature is the differentiation of the
axonal membrane into distinct molecular, structural
and functional domains. These domains include the
nodes of Ranvier, the paranodal junction, the juxtaparanodes and the internodal region8,9 (FIGS 1b and 2).
We will focus on the molecular mechanisms that
underlie the generation and maintenance of these
unique axonal domains, which are necessary for normal
nerve function.
The node of Ranvier
The nodes of Ranvier are short, periodical interruptions
in the myelin sheath, which are spaced at intervals that
are about 100 times the axonal diameter. Although PNS
and CNS nodes show similar structural characteristics,
there are some differences. In peripheral nerves, the
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REVIEWS
a
CNS
Oligodendrocyte
Nerve
terminals
Neuron
cell body
Initial
segment
Axon
Perinodal
astrocyte
PNS
Schwann cell
Nodes
Basal lamina
b
Basal lamina
Microvilli
SpJ
PNS
c
Internode
OMA
IMA
Axon
Internode
JXP
Paranode
Node
d
CNS
PL
Perinodal astrocyte
Juxtamesaxon
PN JXP
Internode
Juxtaincisure
Figure 1 | Structure of myelinated axons. a | Myelinating glial cells, oligodendrocytes in the
central nervous system (CNS) or Schwann cells in the peripheral nervous system (PNS), form
the myelin sheath by enwrapping their membrane several times around the axon. Myelin covers
the axon at intervals (internodes), leaving bare gaps — the nodes of Ranvier. Oligodendrocytes
can myelinate different axons and several internodes per axon, whereas Schwann cells
myelinate a single internode in a single axon. b | Schematic longitudinal cut of a myelinated fibre
around the node of Ranvier showing a heminode. The node, paranode, juxtaparanode (JXP)
and internode are labelled. The node is contacted by Schwann cell microvilli in the PNS or by
processes from perinodal astrocytes in the CNS. Myelinated fibres in the PNS are covered by a
basal lamina. The paranodal loops form a septate-like junction (SpJ) with the axon. The
juxtaparanodal region resides beneath the compact myelin next to the paranode (PN). The
internode extends from the juxtaparanodes and lies under the compact myelin.
c | Schematic cross-section of a myelinated nerve depicting the inner and outer mesaxons
(IMA and OMA, respectively). d | Drawing of the specializations found along the internodes.
A strand composed of paranodal molecules (Caspr, Contactin; red line) flanked by
juxtaparanodal proteins (Caspr2, K+ channels and TAG-1; blue lines) extends along the
internodal region (the juxtamesaxon) and below the Schmidt–Lanterman incisures (the
juxtaincisure). In addition, Nf155 and ezrin–radixin–moesin proteins, as well as connexins 29
and 32 are found at the glial side, opposite these axonal strands.
NATURE REVIEWS | NEUROSCIENCE
entire myelin unit is covered by a basal lamina and
the outermost layer (the outer collar) of the Schwann
cell extends microvilli that cover the nodes (FIG. 1b).
The perinodal space (that is, the space between the
axolemma and the basal lamina), which contains
the microvilli, is also filled with a filamentous matrix10.
In the CNS, there is no basal lamina, and the nodes are
contacted by perinodal astrocytes11,12, recently termed
synantocytes13.
The nodal axolemma. The nodes are characterized by
a high density (>1200/µm2) of Na+ channels that
are essential for the generation of the action potential
during saltatory conduction14. Voltage-gated Na+
channels are multimeric complexes that consist of
a pore-forming α-subunit and one or more auxiliary
β-subunits15 (FIG. 2a). These subunits are encoded
by nine α- (Scn1a–Scn9a) and four β-subunit genes
(Scn1b–Scn4b) in mammals16,17. Nodes of Ranvier in
the adult CNS and PNS mostly contain Nav1.6 (REF. 18).
In addition, Nav1.2 and Nav1.8 are found in many CNS
nodes19, whereas Nav1.9 is localized in some nodes in
the PNS20. During development, both PNS and CNS
nodes express Nav1.2, which is later replaced by Nav1.6
(REFS 21,22). The functional significance of this switch is
currently unclear, but it might allow neurons to adapt
to high-frequency firing23. In addition to voltage-gated
Na+ channels, several other transmembrane and
cytoskeletal proteins have been identified at the nodal
axolemma — the cell-adhesion molecules (CAMs)
of the immunoglobulin (Ig) superfamily Nrcam and
neurofascin-186 (Nf186)24, the cytoskeletal adaptor
ankyrin G25,26 and the actin-binding protein spectrin
βIV (REF. 27). Recent studies have also disclosed the
presence of two K+ channels at the nodes — Kv3.1
(REF. 28) and Kcnq2 (REF. 29). Kv3.1 is mainly found in
large axons in the CNS and only in few nodes in the
PNS, whereas Kcnq2 is located in all PNS nodes and
most CNS nodes.
Na+ channel β-subunits and CAMs. Na+ channel
β-subunits have been shown to modulate channel gating, to facilitate the delivery of Na+ channels to the cell
surface, and to act as CAMs30. The extracellular domain
of these β-subunits has a single Ig domain31, which
mediates homophilic interactions32, as well as binding to
other nodal components. The β1- and β3-subunits
interact in cis with Nf186 (REF. 33), and β1 also binds
contactin34, a glycosylphosphatidylinositol (GPI)
anchored glycoprotein that is found in all paranodes
(see later in text) and in CNS nodes35. The interaction
with contactin enhances the expression of Na+ channels
on the surface of transfected cells, indicating that this
CAM might be important for the expression of Na+
channels at the node of Ranvier34,36. In agreement with
this idea, the expression of these channels is markedly
reduced in the optic nerve of contactin-null mice37. The
β1- and β2-subunits also interact with the extracellular
matrix molecules tenascin-C and tenascin-R38,39, as well
as with phosphacan40, the secreted form of receptor
protein tyrosine phosphatase β (Rptpβ).
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a
b
Schwann/microvilli/
perinodal astrocyte
4.1B
Paranodal loop
ERM
DG
?
Nf186
Nrcam
Tenascin
NG2
Kcnq2
Kv3.1b
Nf155
Caspr
Phosphacan
NaCh
Contactin
Bral1
βαβ
Node
Paranode
MBD
S
Spectrin βIV
c
Protein 4.1B
S
C
C
Compact
myelin /
Schwann
/ Microvilli
Perinodal Astrocyte
d
CNS synapse
Pre
4.1N
PTB
Ankyrin G
Mint
PDZ PDZ
PDZ
GUK
CASK
Ca2+ channels
Connexin 29
Neurexin
Caspr2
Tag1
K+ channel
Neuroligin
NMDAR
GUK
PDZ PDZ PDZ
Psd95
GUK
Protein 4.1B
MBD
S
C
PDZ
Post
PDZ PDZ
PDZ
Psd95
GUK
Cytoskeleton
Juxtaparanode
Figure 2 | Molecular composition of the nodal domains. The specialized domains around the
node of Ranvier are composed of a distinct set of molecules. a | At the nodal axolemma, voltagedependent Na+ channels are anchored to the cytoskeleton by ankyrin G, which also binds Nf186,
Nrcam and Kv3.1b. Ankyrin G connects these proteins to the axonal cytoskeleton through
spectrin βIV. In the PNS, Schwann cell microvilli express ezrin–radixin–moesin (ERM) proteins and
dystroglycan (DG). The nodal gap also contains several extracellular-matrix proteins. NaCh, Na+
channel; NG2, NG2 proteoglycan. b | At the paranodes, a Caspr/contactin complex in the
axolemma faces neurofascin 155 (Nf155) at the glial membrane. Whereas contactin alone can
bind Nf155, Caspr inhibits this interaction, indicating that the Caspr/contactin complex might bind
an unidentified ligand at the glial loops. The cytoplasmic tail of Caspr interacts with protein 4.1B,
providing a potential link with the actin cytoskeleton. c | At the juxtaparanodal axolemma, voltagegated K+ channels are found in a macromolecular complex with Caspr2, protein 4.1B, Psd95 and
Tag1. Tag1 is also expressed on the glial membrane and binds the axonal Caspr2/Tag1 complex.
Connexin 29, localized at the juxtaparanodal glial membrane, could form functional
hemichannels. d | At synapses in the central nervous system (CNS), neurexins interact with
calmodulin-dependent serine kinase (CASK) and the protein Mint, which in turn can associate
with Ca2+ channels. CASK could also bind protein 4.1N, further linking the complex to the actin
cytoskeleton. The extracellular domain of neurexin binds to neuroligin that is present at the
postsynaptic membrane. The cytoplasmic tail of neuroligin interacts with Psd95, which in turn
might recruit NMDA (N-methyl-D-aspartate) receptors (NMDAR). C, carboxy-terminal ;
S, spectrin-binding domain; GUK, guanylate kinase-like domain; MBD, membrane binding domain.
Cytoskeletal proteins. The nodes and the initial segment
are enriched in ankyrin G, a membrane–cytoskeleton
adaptor that links integral membrane proteins to the
spectrin cytoskeleton25,26. Ankyrin G interacts with Na+
channels41, both with their α- (REF. 42) and β- (REF. 32)
970
subunits, as well as with Nf186, Nrcam43 and Kv3.1
(REF. 28). The β-subunit recruits ankyrin G to the plasma
membrane32 and this interaction is regulated by tyrosine
phosphorylation44. The binding of ankyrin G to the
α-subunit is mediated through a sequence of nine
amino acids that is present in all known voltage-gated
Na+ channels42. This nine-amino-acid motif is required
for the accumulation of the α-subunit in the axon initial
segment45. Furthermore, this ankyrin-binding site is
located within a short sequence that is sufficient to target
proteins to the axon initial segment45. It remains to be
determined whether this short sequence is also necessary
for targeting to the nodes of Ranvier. Binding of ankyrin
G to the two nodal Ig-CAMs, Nf186 and Nrcam, is
mediated by a twelve-amino-acid motif that is found in
their cytoplasmic domains43. Ankyrin G binds this motif
only when it is dephosphorylated43,46,47, indicating that
unidentified tyrosine kinases and phosphatases might
regulate this interaction. Tyrosine-phosphorylated
neurofascin is located at the glial paranodes48, but not in
the nodes, supporting the idea that nodal neurofascin is
closely associated with ankyrin G49. Ankyrin G also binds
spectrin βIV, a spectrin isoform that is enriched at the
nodes of Ranvier and axon initial segments27, further
anchoring the nodal Na+ channel and Ig-CAMs to the
axonal cytoskeleton.
| DECEMBER 2003 | VOLUME 4
The nodal gap, extracellular matrix and the glial
membrane. In the PNS, the nodal gap is filled with
Schwann cell microvilli that emanate from the outer
aspect of the cell (FIG. 1b). At the proximal region of the
microvilli, the membranes of two adjoining Schwann
cells are connected by TIGHT JUNCTIONS50,51. However, these
junctions do not seal the nodal gap, as it was found to be
permeable to horseradish peroxidase applied outside
the nerve fibres52. Three proteins — ezrin, radixin and
moesin, as well as the ezrin-binding protein EBP50
and the Rho-A GTPase, are localized at the microvilli53–55.
These proteins might potentially link the actin-rich
microvilli56 with integral membrane proteins57. In addition, several extracellular matrix (ECM) proteins are
present in the nodal gap under the basal lamina, including the hyaluronan-binding proteoglycan versican58,
tenascin-C59,60 and the NG2 proteoglycan61. Recently, it
was shown that dystroglycan, which is abundantly
expressed at the ABAXONAL surface of myelinating Schwann
cells62, is also located at the nodes63. Specific ablation of
dystroglycan in Schwann cells results in the disorganization of the microvilli, a marked reduction in nodal Na+
channels and consequently impaired nerve conduction63.
In contrast to the PNS, processes of perinodal astrocytes contact most of the nodes in the CNS. Here, the
nodal gap has been shown to include several proteoglycans and ECM proteins that are produced by oligodendrocytes, including tenascin64 and phosphacan65.
The CNS nodal gap also contains the versican-binding
protein Bral1, which is produced by neurons66. The
function of these proteins is presently unclear, although
it was suggested that, owing to their high content
of acidic disaccharides, they could provide a strong
negative environment that serves as an extracellular
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Box 1 | Vertebrate axoglial junctions and Drosophila septate junctions
a
b
Membrane
Caspr
Caspr2
Nrx4
Neurexin-4
The axoglial paranodal junction
Neurexin-1a
Neuroligin
Gliotactin
Nrg
c
Contactin
Tag1
Dcon
Dcon
Nf155
Neuroglian
DISC
FNIII
LamG
Ig
EGF
FIB
Lectin
4.1B
PDZ-B
AChE
Nrg
In Drosophila, a blood–brain barrier is formed by perineural and glial cells, which insulate
neurons from the surrounding hemolymph and allow the normal propagation of action
potentials. Septate junctions that are present between these cells are necessary for the
integrity of this blood–brain barrier81,166–168. Septate junctions, which are also found in all
invertebrate epithelia, share morphological, functional and molecular similarities with the
vertebrate paranodal junction. Both junctions contain regularly spaced electron-dense
septa that give them a ladder-like appearance. Disruption of septate junction or paranodal
junction integrity results in abnormalities in the propagation of axonal action
potentials81,86,87,90,166. The basic molecular components of the fly septate junctions seem to
be conserved in the vertebrate paranodal junction (a). Paranodal junctions contain a
complex of Caspr and contactin35, whereas neurexin-4 (Nrx4), the fly homologue of Caspr
and Caspr2, is found in Drosophila septate junctions81, where it co-localizes and interacts
with Drosophila contactin (Dcon) (b–c; M. Shelly and E.P., unpublished observations).
Both Caspr and Nrx4 associate with 4.1 proteins (4.1B and Coracle, respectively), which
stabilize them at the junction88,101. Drosophila septate junctions also contain the Nf155homologue neuroglian, which associates with the Nrx4/Coracle complex167. The figure
shows staining of wild-type Drosophila embryos (stage 11–15) with the indicated
antibodies; Nrx4, Dcon and neuroglian co-localized at cell junctions of the outer
ectoderm. Two other proteins, gliotactin and a Na+/K+ ATPase, are localized at septate
junctions, and are important for their formation167,168, but their mammalian homologues
have not been reported to reside in the axoglial paranodal junction. Panels b–c show the
expression of Drosophila homologues of the paranodal junction components in the fly
epithelia. AChE, acetylcholinesterase; DISC, discoidin-like domain; EGF, epidermal
growth factor; FIB, fibrinogen-like domain; FNIII, fibronectin-III-like domain;
Ig, immunoglobulin-like domain; LamG, laminin G; PDZ-B, PDZ-binding domain.
TIGHT JUNCTION
A belt-like region of adhesion
between adjacent cells. Tight
junctions regulate paracellular
flux, and contribute to the
maintenance of cell polarity by
stopping molecules from
diffusing within the plane of the
membrane.
ABAXONAL
Term that refers to the outermost
layer of the myelin sheath.
TYPE I TRANSMEMBRANE
PROTEIN
Molecule with a single
transmembrane domain.
CNS myelinated axons are normal in Rptpβ-deficient
mice68. Notably, both tenascin-R and Rptpβ also interact
with contactin and Nrcam69–71, which are present at
CNS nodes, indicating the possible existence of large
macromolecular complexes at the perinodal space.
Na+ reservoir in the perinodal space66. Both tenascin-C
and tenascin-R bind to Na+ channels39 and alter their
electrophysiological properties38. Genetic ablation of
tenascin-R resulted in slower nerve conduction, but had
no effect on the distribution of Na+ channels at the
nodes, indicating that this interaction might stabilize
nodal complexes or regulate channel activity, but is not
required for the initial clustering of these channels67. Na+
channels were also reported to bind the cytoplasmic tail
and the extracellular domain of Rptpβ40, a receptor
tyrosine phosphatase that has not been reported to be
located at the nodal axolemma. Furthermore, the
importance of these interactions for the normal physiology of myelinated nerves is not clear, as the distribution
of nodal Na+ channels and the conduction velocity of
NATURE REVIEWS | NEUROSCIENCE
Morphology and molecular composition. At both sides of
the nodes of Ranvier, the compact myelin membrane
opens up and forms cytoplasm-filled glial loops that
wind helically around the axon (FIG. 1b). These paranodal
loops are connected to the axolemma by a series of ridges
(transverse bands) that are reminiscent of invertebrate
septate junctions72 (BOX 1). The axoglial junctions appear
relatively late during myelination, being first generated
closer to the nodes by the outermost paranodal loop, and
continue gradually as additional loops are attached to the
axon73. As a result, they are composed of a number of
rings, each representing a turn of the myelin wrap.
The axonal membrane at the axoglial junction
contains a complex of two cell-recognition molecules —
contactin-associated protein (Caspr; also known as
paranodin)74,75 and contactin35 (FIG. 2b). Caspr is a TYPE I
TRANSMEMBRANE PROTEIN that belongs to a distinct subgroup
of the neurexins, a polymorphic protein family that is
involved in cell adhesion and intercellular communication76,77. There are five human genes in the Caspr family
(CASPR1–CASPR5 (REFS 78–80)), two in Drosophila81,82
(nrxIV and axo) and two in Caenorhabditis elegans
(itx-1 and nlr-1; L. Haklai-Topper and E.P., unpublished
observations). These proteins bind several CAMs and
should therefore be considered as CAM-associated
proteins. Their extracellular region consists of several
domains that are implicated in protein–protein interactions, including a discoidin and a fibrinogen-like domain,
epidermal growth factor (EGF) motifs, and several
regions with homology to the G domain of laminin A
(BOX 1). Caspr, but not other members of the Caspr family, forms a complex with contactin only in CIS78. The
interaction between Caspr and contactin is required
for the efficient export of Caspr from the endoplasmic
reticulum to the plasma membrane83, and regulates the
glycosylation and transport of contactin84. Caspr and
contactin are associated in the endoplasmic reticulum
and might be transported through a Golgi-independent
pathway to the cell surface84,85. In agreement with these
in vitro findings, Caspr is retained in the neuronal
somata and does not reach the axons in contactindeficient mice86, whereas Caspr is necessary to maintain
contactin at the paranodes84,87,88.
Both Caspr and contactin are essential for the generation of the axoglial junction, and their absence results in
the disappearance of septa and a widening of the space
between the axon and the paranodal loops84,86,87. These
results indicate that Caspr and contactin might be part of
a paranodal adhesion complex that is required for the
tight attachment of the two membranes. This phenotype
is similar to those of two other paranodal mutants: the
galactolipids-deficient mice, which lack UDP-galactose
ceramide galactosyltransferase (Cgt) and do not
synthesize galactocerebroside (GalC) and sulfatide,
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CIS INTERACTION
Term that refers to the
interaction between molecules
that are present in the same cell
membrane, as opposed to an
interaction in trans, in which the
interacting molecules are present
in opposing membranes.
LIPID RAFTS
Dynamic assemblies of
cholesterol and sphingolipids in
the plasma membrane.
MULTIPLE SCLEROSIS
A neurodegenerative disorder
characterized by demyelination
of central nervous system tracts.
Symptoms depend on the site of
demyelination and include
sensory loss, weakness in leg
muscles, speech difficulties, loss
of coordination and dizziness.
FREEZE FRACTURE
An electron-microscopic method
in which rapidly frozen tissue is
cracked to produce a fracture
plane through the specimen. The
surface of the fracture plane is
shadowed by a heavy metal, and
the specimen is digested away to
leave a replica that can be
examined under the electron
microscope.
972
and cerebroside sulfotransferase (Cst)-null mice, which
only lack sulfatide89–93. In all of these mutants, Caspr and
contactin are absent from the paranodes86,87,93–95.
The way in which the absence of GalC and sulfatide
causes paranodal abnormalities is not clear, but
it might result from direct binding of sulfatide to
the Caspr/contactin complex. Alternatively, given the
proposed role of galactolipids in the formation of
96,97
LIPID RAFTS and the organization of myelin
, their
absence might result in a misrouting of junctional glial
components to non-compact myelin. The latter possibility is further supported by recent findings, showing
that genetic ablation of the myelin and lymphocyte
(MAL) protein, a raft-associated molecule that is
involved in intracellular trafficking, results in paranodal
abnormalities (N. Schaeren-Wiemers and U. Suter,
personal communication).
The intracellular regions of Caspr and Caspr2
contain a juxtamembrane sequence that binds protein
4.1B75,78,88,98, which is present at the paranodes and juxtaparanodes95,98–100. Similar to other 4.1 proteins, 4.1B
contains a conserved actin–spectrin-binding domain
and could therefore immobilize Caspr (and therefore
contactin) to the cytoskeleton88. Consistent with this
idea, protein 4.1B is abnormally distributed along
peripheral myelinated axons of mice lacking either
contactin or galactolipids, both of which lack paranodal
Caspr88,95. In these mutants, the position of protein 4.1B
correlates strongly with those of Caspr and Caspr2,
indicating that they might determine its localization.
Furthermore, the cytoplasmic tail of Caspr is required
for stabilizing the Caspr/contactin complex at the paranodes, as a Caspr mutant that lacks this domain is not
properly maintained at the axoglial junction88. So, Caspr
seems to serve as a transmembrane scaffold that stabilizes the Caspr/contactin adhesion complex at septatelike junctions by connecting the complex to the axonal
cytoskeleton through protein 4.1B. This mechanism
closely resembles the function of Drosophila neurexin IV,
which recruits Coracle (the homologue of protein 4.1) to
septate junctions81,101 (BOX 1). In addition, the cytoplasmic
region of Caspr also binds the FERM domain (fourpoint-one, ezrin–radixin–moesin)-containing protein
Schwanomin/merlin102. However, the importance of this
interaction is less clear, as Schwanomin is not concentrated at the paranodal junction.
The distribution of Caspr and contactin along the
internodes95,103,104 (see later in text), their accumulation at
the paranodes as a number of rings that represent each
turn of the myelin wrap during development35,105,106, and
the abnormal distribution of Caspr in MULTIPLE SCLEROSIS107
and in several myelin mutants19,93–95,105,108 indicate that
the myelin sheath dictates the localization of Caspr and
contactin in the axolemma. Furthermore, the addition of
a soluble Rptpβ, which binds contactin, to myelinating
co-cultures perturbs the paranodal accumulation of
Caspr, indicating that the localization of the Caspr/
contactin complex to this site might be mediated by
its interaction with a glial ligand35. The most probable
candidate to serve as a glial ligand of the Caspr/
contactin complex is Nf155, a glial isoform of the CAM
| DECEMBER 2003 | VOLUME 4
neurofascin, which is located across Caspr and contactin
at the axoglial junction48, and is not localized to this site in
the absence of Caspr86,87,95,109. In agreement with this idea,
it was recently reported that a soluble Nf155-Fc chimaera
binds to cells that express Caspr and contactin, and precipitates these proteins from rat brain lysates, indicating
that Nf155 might indeed serve as a receptor for the
Caspr/contactin complex110. However, recent studies have
challenged this model, showing that, whereas Nf155
binds directly to contactin, Caspr inhibits this interaction.
This observation indicates the possible existence of other
receptors for the Caspr/contactin complex in myelinating
glia84. This conclusion agrees with previous observations
that show that Nf155 appears much later than Caspr in
the paranodes109.
Function of the paranodal junction. The paranodal junction was proposed to attach the myelin sheath to the
axon, to separate the electrical activity at the node of
Ranvier from the internodal region under the compact
myelin sheath, and to serve as a fence that limits the lateral diffusion of axolemmal proteins111. Recent studies
using four different paranodal mutant mice — mice
lacking Caspr, contactin, Cgt and Cst, all of which lack the
characteristic septa in their axoglial junction — allowed
close examination of these original ideas. In the CNS of
these mutants, the paranodal loops are disorganized,
with many overlapping and inverted loops that face away
from the axon87,92,112. In the PNS, the morphological
alterations are much milder, possibly due to the presence
of the basal lamina; the paranodes are well organized,
but there is an increase in the space between the glial
membrane and the axon. However, even in the absence
of septa, the paranodal loops are still closely attached to
the axon in many sites in the PNS and CNS, pointing
to the presence of so far unidentified paranodal components that mediate axoglial contact at this site. Together
with ultrastructural data showing that the transverse
bands are generated late during myelination73,109, these
studies indicate a possible role for the septa in securing
the paranodal loops to the axon at the axoglial junction.
In agreement with this view, a gradual, age-dependent
detachment of the paranodal loops from the axon was
observed in the CNS of Caspr-null mice104.
The absence of paranodal septa in all four paranodal
mutants results in a reorganization of the axonal membrane86,87,93–95 (FIG. 3). In these mutants, the shaker-type
K+ channels that are normally present in the juxtaparanodal region are mislocalized to the paranodal axon
membrane86,87,93–95. So, it seems that the paranodal
septate junction functions as a barrier that restricts the
movement of K+ channels from under the compact
myelin, separating them from the Na+ channels at the
nodes. In contrast to the juxtaparanodal K+ channels,
disruption of the paranodal septa minimally affects the
distribution of the nodal Na+ channels86,87,94. There is a
small increase in nodal length, accompanied by a reduction in membrane particles at the nodal axolemma, that
is detected by FREEZE-FRACTURE electron microscopy, indicating that the paranodal septate junction might not
be required for the generation of the nodes87,104,113.
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Wild type
Juxtaparanode
Paranode
Caspr2
Tag1
Node
Dystroglycan
Spectrin βIV
Caspr
Contactin
Cgt
Cst
Figure 3 | Arrangement of the nodal environ in various mutant mice. A schematic
representation of the paranodal loops attaching to the axon. The distribution of nodal (red),
paranodal (purple) and juxtaparanodal (blue) proteins is shown in wild-type and the indicated
mutant mice. In paranodal mutants (Caspr, contactin, ceramide galactosyltransferase (Cgt) and
cerebroside sulfotransferase (Cst), all juxtaparanodal components move to the paranodes; in
juxtaparanodal mutants (Caspr2 and Tag1), K+ channels are dispersed along the internodes; in
nodal mutants (dystroglycan and spectrin βIV), Na+ channel clustering at the nodes is reduced.
See TABLES 1 and 2 for a detailed description of these and other mutants.
However, glial attachment at the paranodes in the CNS is
required to maintain Na+ clustering at the nodal
axolemma93,104,113,114.
Juxtaparanodal specialization
DELAYED RECTIFIER
K+ CHANNELS
Slowly activating and very slowly
inactivating channels that
preferentially pass K+ out of
the cell.
PDZ DOMAIN
A peptide-binding domain that is
important for the organization of
membrane proteins, particularly
at cell–cell junctions, including
synapses. It can bind to the
carboxyl termini of proteins or
can form dimers with other PDZ
domains. PDZ domains are
named after the proteins in which
these sequence motifs were
originally identified (PSD95,
Discs large, zona occludens 1).
ADAXONAL
Term that refers to the innermost
layer of the myelin sheath.
GAP JUNCTIONS
Cellular specializations that allow
the non-selective passage of small
molecules between the cytoplasm
of adjacent cells. They are formed
by channels termed connexons
— multimeric complexes of
proteins known as connexins.
Gap junctions are structural
elements of electrical synapses.
The juxtaparanode is located in a short zone just beyond
the innermost paranodal junction (FIG. 1b). In freezefracture electron microscopy, this region shows randomly
distributed particles that are more concentrated near the
paranodes and diffuse away towards the internodes111.
These particles most likely correspond to heteromultimers of the DELAYED RECTIFIER K CHANNELS of the Shaker
family, Kv1.1, Kv1.2 and Kvβ2 (REFS 115,116). At the juxtaparanodal axolemma, these channels co-localize and
create a complex with Caspr2, the second member of the
Caspr family79. In addition, Kv1.6 is present at this site,
predominantly in small axons117. Two other proteins that
are found at the juxtaparanodes are transient axonal
glycoprotein-1 (Tag1), a GPI-anchored CAM that is
related to contactin118, and connexin 29 (Cx29), which is
found at the glial membrane119,120. The association
of Caspr2 with K+ channels is mediated by their carboxyterminal region, most probably through an unidentified
PDZ DOMAIN-containing protein. Although one such
protein, Psd95, is located at the juxtaparanodes and
associates with K+ channels, it does not mediate
the interaction of these channels with Caspr2 or their
accumulation at this site121,122. Two recent studies showed
that Caspr2 and Tag1 form a juxtaparanodal complex,
consisting of a glial Tag1 molecule and an axonal
Caspr2/Tag1 heterodimer123,124 (FIG. 2c). This complex
is essential for the accumulation of K+ channels in the
juxtaparanodes, as targeted disruption of Caspr2 or Tag1
results in a striking reduction in the juxtaparanodal
accumulation of these channels in both PNS and
CNS axons (FIG. 3). These results indicate that Caspr2
and Tag1 might form a scaffold that enables the
+
NATURE REVIEWS | NEUROSCIENCE
positioning of ion channels at specific sites of the plasma
membrane, therefore resembling the mechanisms that
operate during synapse formation (FIG. 2d).
Role of K+ channels under the myelin sheath. Juxtaparanodal K+ channels were proposed to act as an active
damper of re-entrant excitation and to help in maintaining the internodal resting potential125–128. Although
theoretically it is enough to have these channels scattered
along the internodes to maintain the resting potential,
preventing re-entrant excitation would require a high
spatial clustering of K+ channels near the node. Despite
the marked abolishment in juxtaparanodal clustering of
Kv1.1/Kv1.2 in Caspr2- and Tag1-knockout mice, there is
no change in the excitability of myelinated nerves123,124.
The observation that the total content of these channels
remains constant in both mutants could indicate that the
main role for these myelin-concealed K+ channels is
maintaining the internodal resting potential. In addition,
a computer model in which both K+ channel distribution
and the axoglial junctional conductance were varied
indicated that the clustering of K+ channels in the juxtaparanode could provide a protective function in axons
that might undergo a low degree of demyelination (FIG. 4).
A testable implication of this model is that Caspr2
and Tag1 might serve to ensure stability in axons with
compromised axoglial junctions.
Another function of the juxtaparanodal K+ channels
might be mediating axoglial communication. In the
PNS, these channels are located across from Cx29
hemichannels that are present at the ADAXONAL membrane
of myelinating Schwann cells119,120, which most likely
correspond to the rosettes of particles that are seen by
freeze-fracture electron microscopy at this site129. These
hemichannels could provide a direct pathway for K+ ions
from the axon to the overlying glia119. This, in turn,
would generate an activity-dependent signal into the
Schwann cell, reminiscent of electrical synapses formed
by GAP JUNCTIONS. In support of this idea, Ca2+ transients
recorded in Schwann cells upon electrical stimulation of
the axon were proposed to be generated by K+ efflux
from the axon that depolarizes the glial membrane130.
The exchange of information through such an ‘axoglial
synapse’ at the juxtaparanodes could provide an additional mechanism for axon–gliacommunication131.
Interestingly, the paranodal axoglial junction could also
be remodelled by neuronal activity132, an effect that could
be mediated, in part, by controlling the expression of
contactin on the axonal surface133.
Internodal differentiation
Although no junctional specializations are observed
between the glia and the axon along the internode,
freeze-fracture electron microscopy revealed that the
internodal axolemma in the PNS contains longitudinal
strands of intramembranous particles that resemble
those found in the paranodes and juxtaparanodal
region134,135. As shown in FIGS 1c,d, Caspr and contactin
are located throughout the internodal region in a strand
that is flanked by K+ channels and Caspr2, which apposes
the inner mesaxon of the myelin sheath and forms a
VOLUME 4 | DECEMBER 2003 | 9 7 3
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Formation of the nodal environ
25 mV
5 ms
a
Internode
Juxtaparanode
b
Paranode
c
Node
-araP
edon
Paranode
PXJ
Juxtaparanode
Internode
d
Figure 4 | A computational model describing a role for juxtaparanodal K+ channels in
myelinated fibres. Each part of the figure shows a schematic organization of the paranodal
junction (black lines) and the distribution of K+ channels (purple ovals), together with the
corresponding action potential recorded after a single stimulus. a | The axon has normal
properties and responds to a single stimulus with a single action potential. b | The paranodal
junctions remain normal, but K+ channel clusters have dissipated into the internode (Caspr2 –/–;
Tag1–/–) and conduction velocity remains normal. c | The junctions are loosened to increase
conductance to 10% of the value that is observed when they are fully open, but the channels
remain clustered. Conduction velocity is slowed by 40%, but is otherwise stable. d | The junctions
are loosened as in c, but the channels are now dispersed as in b, and the axon responds to a
single stimulus with repetitive action potentials. The model was kindly provided by P. Shrager and
used parameters that have been described by Hines and Shrager169.
SCHMIDT–LANTERMAN
INCISURE
A cytoplasmic channel that
interconnects the adaxonal and
abaxonal layers of the myelin
sheath.
LAMINA CRIBROSA
The supporting structure for the
optic nerve at the point in which
it leaves the eye.
974
circumferential ring just below the inner aspect of the
35,95,103
SCHMIDT–LANTERMAN INCISURES
. This line, termed juxtamesaxonal and juxta-incisural9,136, is a direct continuation
of the paranodes/juxtaparanodes. Accordingly, Nf155
(REF. 48), Cx29 (REF. 119) and Tag1 (REF. 118) are localized
in a complementary distribution on the adaxonal membrane of myelinating Schwann cells. These findings indicate that the internodal localization of axonal proteins is
dictated by the myelin sheath, probably by mechanisms
similar to those that operate in the paranode/juxtaparanode. However, recent analysis of Caspr2-null mice
indicates that different mechanisms might control the
localization of K+ channels in the juxtaparanodes and
the juxtamesaxon124. The molecular organization of the
internodal region is not observed in myelinated nerves in
the CNS48,118,119,137.
| DECEMBER 2003 | VOLUME 4
The role of myelinating glia. During the development of
myelinated nerves in the PNS, the different nodal
domains are formed gradually; Na+ channels are first
clustered at the nodes, followed by the generation of the
paranodal junction, and later on by the clustering of K+
channels at the juxtaparanodal region22,125,138. In both the
CNS and the PNS, Na+ channels cluster initially at sites
that are adjacent to the edges of processes extended by
oligodendrocytes22,105 and myelinating Schwann cells138,139.
Further longitudinal growth of these processes causes
displacement of the clusters until ultimately two neighboring clusters seem to fuse, forming a new node of
Ranvier. These results indicate that these Na+ clusters are
positioned by direct glial contact.Accordingly, the distribution of Na+ channels is diffuse along retinal ganglion
cells, but they are clustered at the nodes right after these
axons cross the LAMINA CRIBROSA and become myelinated22.
These channels are not clustered after ablation of oligodendrocytes140 or Schwann cells138, and are dispersed
during demyelination141. Furthermore, nodal Na+ channels are associated with the edges of myelinating
Schwann cells in nerves that display shorter internodes as
a result of remyelination141 or genetic mutation, as seen in
the CLAW PAW mutant mouse142. However, studies using
retinal ganglion cells showed that Na+ clustering could be
induced in vitro by soluble factors that are secreted by
cultured oligodendrocytes21,143. Although Schwann cells
do not secrete such clustering activity139, some clustering
of Na+ channels has been detected in the absence
of myelinating Schwann cells in dystrophic mice144.
Recent analyses of dysmyelinating145,146 or paranodal
mutants104,147, and models of demyelination114,148 showed
that the presence of intact myelinating oligodendrocytes
is also required for the developmental switch of Na+
channel isoform in the nodes. By contrast, Nav1.6 is
found in the nodes of two myelin mutants that are associated with oligodendrocyte death and lack normal paranodal junctions — MYELIN DEFICIENT (MD) RATS and JIMPY
mutant mice. This observation indicates that the switch
might occur in the absence of normal paranodal contact
or myelin19,108. Notably, recent analysis of the SHIVERER
mutant revealed that, whereas axoglial contact is necessary for the expression of Nav1.6 at nodes, it is not
required for targeting of this subunit to the axon initial
segment, pointing to the existence of multiple targeting
mechanisms in myelinated axons149.
Molecular assembly. During the development of myelinated nerves in the PNS, Nrcam and Nf186 are detected
at the nodes first, followed by the appearance of ankyrin
G and Na+ channels150. In the CNS, however, ankyrin G is
detected at the nodes before the clustering of Nf186 and
Na+ channels108. These results indicate that Nrcam, Nf186
or an unidentified ankyrin G-binding protein binds
ankyrin G, which in turn recruits Na+ channels. In support of this model, the addition of a soluble Nrcam to
myelinating dorsal root ganglia cultures inhibits Na+
channel clustering151. Moreover, the appearance of
ankyrin G and Na+ channels at the nodes is delayed
in Nrcam-null mice152, indicating that this adhesion
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Table 1 | Molecular changes at the nodal region in myelin-mutant mice
Mutant/gene
Node
Paranodes
Juxtaparanodes
shiverer
Mbp mutant
(CNS hypomyelination)
(slight PNS
hypomyelination)
Fewer Na+ channel clusters;
most are atypical. No Na+
channel isoform switch. Expression
of Na+ channel is elevated.
Rare Nav1.6 clusters adjacent to
Caspr-labelled zones, but normal
clusters in the initial axon segment.
No ankyrin G clustering
Axoglial junction abnormalities;
aberrant location. Irregular
Caspr/Nf155-labelled patches.
Caspr next to the few existing
nodes
Adult CNS: Kv1.2 not clustered;
22,48,105,
diffusely distributed; present
117,149,
adjacent to few normal nodes.
170
Increased overall expression of Kv1.2.
PNS: mildly affected. Slight increase
in internodal staining and occasional
elongated juxtaparanodes
References
trembler
Pmp22 mutant
(PNS hypomyelination)
Clusters of ankyrin G, Na+
channels and Nf186; some
binary clusters
Terminal loops face outwards
in some fibres
Kv1.1 is redistributed along the axon
150,179,
171
Plp overexpression
(CNS demyelination)
Gradual decrease of Na+ channel
clusters as demyelination
progresses; irregular elongated
and binary clusters; decrease in
Nav1.6 clusters and increase in
the total expression of Nav1.2
Marked reduction of paranodal
staining of Caspr in optic
nerve
K+ channels decreased markedly
with age. Eventually all K+ channel
clusters disappear. Total protein level
of K+ channels is unaltered
114,121,
145
jimpy
Plp mutant
(oligodendrocyte death)
(CNS hypomyelination)
Reduced number of nodes;
abnormal shape; binary, broad or
dot-like. Clusters of ankyrin G,
Na+ channels and Nf186. Normal
Na+ channel isoform switch
Disrupted. Caspr absent
(diffusely distributed). No
paranodal ankyrin G is
detected during development
Transient clustering of Kv1.1 in
paranodes, which disappear after
3 weeks
108,121,
140,145
Myelin-deficient (md)
rats.
Plp mutant
(CNS dysmyelination)
Na+ channel and ankyrin G
clusters but many do not
surround the full circumference
of the axon. Normal Na+ channels
isoform switch. Kv3.1b clusters
at nodes
No septate junctions
Absence of Caspr, contactin
and Nf155; Caspr is diffusely
distributed in CNS axons. Total
Caspr and contactin protein
levels are unaffected
Kv1.1 and Kv1.2 in paranodes; some
nodal staining is detected
P0 null
Normal Na+ channels clusters
(Nav1.6); some broad and binary
clusters in adult; larger nodal
gap; aberrant microvilli; shorter
internodes. In contrast to WT
mice, nodes along the femoral
quadriceps motor nerve
expresses Nav1.8
Caspr is either asymmetrically
present in heminodes (53%),
absent (5%) or normal (42%).
Normal Caspr is correlated
with absence of Nav1.8,
representing morphologically
normal nodes
Asymmetric distribution of Kv1.2 in
paranodes; absent in 29% of sites,
Caspr2 is shifted or expanded to the
paranodes; absent in only 7% of sites
E-cadherin null
(Schwann-cell specific)
Normal Na+ channel distribution
Caspr present at paranodes;
normal paranodes.
Normal Kv1.1 and Kv1.2 clusters
Mag null
Na+ channels clustering is not
affected
Caspr and Nf155 staining less
defined, diffused along the
processes. Partial delay in the
formation of septa. More
pronounced paranodal loop
disorganization in Mag/Cgt
nulls than in each mutant alone
Caspr2 absent from juxtaparanodes
Kv1.1 extends to the paranodal
region but is normally localized in
the adult
109,174
Dystrophic
Laminin α2
Short internodal lengths; heminodes. No axoglial septa in ventral
Presence of Na+ channel and
root. Normal Caspr in sciatic
ankyrin G clusters in amyelinated
nerve
axons; in many cases more
extended than WT nodes
ND
144,175
19,28,
143
146,172
173
CNS, central nervous system; Cgt, ceramide galactosyltransferase; Mag, myelin-associated glycoprotein; Mbp, myelin basic protein; ND, no data; Nf, neurofascin;
Plp, myelin proteolipid protein; Pmp, peripheral myelin protein; PNS, peripheral nervous system; WT, wild type.
CLAW PAW
Mutant mice in which peripheral
myelination is disrupted, but
central myelination is unaffected.
The responsible gene has not
been identified.
MYELIN-DEFICIENT RATS
Strain which the gene for the
proteolipid protein is mutated,
leading to defective myelination,
tremors, ataxia and early death.
ataxia, tremor and cerebral
atrophy.
molecule participates in clustering. The eventual formation of nodes in these animals could be explained by the
presence of Nf186, which contains a similar ankyrin
G-binding site and could therefore compensate for the
absence of Nrcam. The importance of the interaction
between ankyrin G and these nodal components was
shown in mice lacking the cerebellar isoform of ankyrin
G, in which Na+ channel, Ig-CAMs and spectrin βIV are
not clustered in the initial segment of Purkinje cell
axons153,154. Similarly, spontaneous mutations of spectrin
βIV in the QUIVERING mice155, or targeted disruption of
this gene156, results in nodal abnormalities and altered
channel distribution. However, ankyrin G is also present
NATURE REVIEWS | NEUROSCIENCE
at the paranodes during the early development of myelinated axons, indicating that it might not be directly
involved in the initial targeting of Na+ channels to the
nodes, but rather be important for their stabilization105,108.
Furthermore, ankyrin G is normally localized at the
nodes in dystroglycan-null mice, which display a marked
reduction of nodal Na+ channel clusters63. After the initial
clustering of nodal components in PNS fibres, Nf155
and the Caspr/Contactin complex accumulate in
the paranodal junction22,35, followed by the arrival of
Caspr2 and K+ channels to the juxtaparanodal
region95,125. Caspr2, K+ channels and TAG-1 are first
detected at the paranodes, and subsequently relocate
VOLUME 4 | DECEMBER 2003 | 9 7 5
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Table 2 | Molecular changes in nodal-environ mutants
Mutant/gene
Node
Paranodes
Juxtaparanodes
References
+
Cgt null
(Paranodal)
CNS: Elongated nodes, abnormal
shape; heminodes; some clusters
do not surround the full axonal
circumference. PNS: minor
expansion of Na+ channel clusters
and ankyrin G. Age-dependent
decrease in the number and
intensity of Nav1.6 cluster;
increased nodal length
Absence of transverse bands.
Caspr and contactin almost
completely absent from
paranodes; diffused staining of
Caspr adjacent to narrow
labelling of paranodal K+
channels; reduced paranodal
labelling of Nf155. Reduced
accumulation of paranodal 4.1B
K channels, Caspr2 and Tag1 are
found in the paranodes in the PNS
and are diffused along the internode
in the CNS; some paranodal
concentration of K+ channels is
observed in spinal cord
94,95,112,
113,118,147
Contactin null
(Paranodal)
PNS: normal appearance of Na+
channels. CNS: fewer and
elongated Na+ channel-labelled
nodes
Absence of transverse bands.
Absence of Caspr from
paranodes (found in soma);
reduced paranodal Nf155.
4.1B is diffusely distributed
along the axon
PNS: K+ channels and Caspr2 are
found in the paranodes
37,86,88,95
Caspr null
(Paranodal)
Elongated nodes (labelled with
Na+ channels, Nrcam and
spectrin βIV); CNS nodes
progressively disperse; normal
ERM positive microvilli. Aberrant
Na+ channel isoform switch in
CNS; switch is delayed in the PNS.
Increased contactin in CNS nodes
Absence of transverse bands.
Absence of contactin and
Nf155 from paranodes.
Progressive detachment of
paranodal loops in CNS
K+ channels and Caspr2 are found
at the paranodes; more diffuse in
the CNS than in the PNS. Kv1.1
clusters are lost over time in the CNS.
Increased juxta-incisural lines along
internodes
84,87,104
Cst null
(Paranodal)
CNS: elongated nodes, abnormal
shape and intensity; binary
clusters. Decreased clustering
with age (12% at 22 weeks)
Disrupted axoglial junction.
Caspr diffusely distributed
along the axon
CNS and PNS: diffuse K+ channels
and PSD-95 with some concentration
at paranodes. Decreased clustering
with age (8% at 22 weeks)
92,93
Caspr2 null
(Juxtaparanodal)
Normal appearance
Normal appearance
Reduced K+ channels clustering.
Absence of TAG-1. Intense
juxtamesaxonal labelling of K+
channels
Tag1 null
(Juxtaparanodal)
Normal appearance
Normal appearance
Reduced K+ channels clustering.
Absence of Caspr2. Small decrease
in juxtaparanodal labelling of 4.1B
Dystroglycan
(Nodal)
Reduced Na+ channel clustering
(90%); channels dispersed along
a broader region (7%). Normal
distribution of ankyrin G, moesin
and Nf186. Abnormal microvilli
morphology
Normal localization of Nf155
Normal localization of K+ channels
and Caspr2
quivering
Spectrin βIV
(Nodal)
ND
ND
Kv1.1 is upregulated and
redistributed along the length of
the axon
155
Spectrin βIV
-genetrap
(Nodal)
55% reduction in number of
nodes as measured by Nav1.6
immunoreactivity. Reduced
intensity of Na+ channels
compared with WT
ND
ND
156
Nrcam null
(Nodal)
Delayed Na+ channel and
ankyrin G clustering in PNS
Normal Caspr localization
ND
152
Na+ channel β2 null
(Nodal)
Normal appearance; Nav1.6
appears within a normal time
course. Reduced Na+ current in
optic nerve; CAP data consistent
with loss of nodal Na+ channels
Normal Caspr localization
ND
176
124
123,124
63
CNS, central nervous system; CAP, compound action potential; Cgt, ceramide galactosyltransferase; Cst, cerebroside sulfotransferase; ERM, ezrin/radixin/moesin; ND, no
data; Nf, neurofascin; Nrcam, neuronal cell-adhesion molecule; PNS, peripheral nervous system; Tag1, transient axonal glycoprotein-1; WT, wild type.
JIMPY
A mouse strain in which the gene
for the proteolipid protein is
mutated, leading to defective
myelination and oligodendrocyte
death.
976
to the juxtaparanodes as the paranodal junction
forms95,123,125,157. In the absence of this junction, K+
channels do not move to the juxtaparanodes and remain
adjacent to the nodes86,87,93–95 (FIG. 4 and, for further
information, see TABLES 1 and 2). Further maintenance of
K+ channels at the juxtaparanodal region requires Caspr2
and Tag1, as these channels are redistributed along the
internodes in their absence123,124.
| DECEMBER 2003 | VOLUME 4
Molecular sieves, pickets and fences. The segregation of
proteins to distinct domains in neurons is achieved
through specific sorting mechanisms, followed by the
anchoring and clustering of these proteins in the plasma
membrane. The formation of the nodal environ might
involve several distinct molecular mechanisms (FIG. 5).
The exclusion of Na+ channels from the extending edges
of myelinating glia during development might be
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a Na+ channels
Node
b Adhesion
c Exclusion
d Selective transport and
endocytosis
Figure 5 | Possible mechanisms involved in node
formation. a | Schematic presentation of the appearance of
Na+ channels during development. Contacting processes from
myelinating glial cells induce the clustering of molecules at the
underlying axonal membrane. This clustering might be mediated
by several distinct mechanisms (b–d), which could operate alone
or in concert (see text for details). b | Na+ channels are directed to
the nodes by adhesive interactions with Schwann cell microvilli
or perinodal astrocytes. c | Na+ channels are excluded from the
paranodes either by a molecular sieve or by a repulsive signal
that is present at this site. d | Clustering of Na+ channels at the
nodes could be achieved by specific transport coupled with
selective endocytosis along the internodes.
SHIVERER
A mouse strain in which the gene
for myelin basic protein is
mutated, leading to a defect in
myelination. These animals are
characterized by the presence of
ataxia, tremor and cerebral
atrophy.
QUIVERING
A mouse strain in which the gene
for spectrin βIV is mutated,
leading to progressive ataxia,
tremor, hindlimb paralysis and
deafness.
mediated by a selective molecular filter111 or sieve106 that is
found at the paranodes (FIG. 5c). It was proposed that such
a sieve selectively excludes large protein complexes,
including Na+ channels and Ig-CAMs that are connected
to ankyrin G, while allowing the passage of small membrane particles, such as those that correspond to K+
channels106,111. This process requires axoglial contact, but
is not mediated by the Caspr/contactin complex, as its
absence does not prevent Na+ channels from clustering at
the nodes86,87. So, the generation of mature, septa-containing paranodal junctions might not be required for the
efficient clustering of Na+ channels. This conclusion is
further supported by freeze-fracture electron microscopic
studies, disclosing an early differentiation of the nodes
prior to the generation of the paranodal septa73. This
implies that the initial axoglial contact at the paranodes
is required for node formation, independently of the
generation of septa. Accordingly, the accumulation of
NATURE REVIEWS | NEUROSCIENCE
Caspr at the paranodes and the nodal clustering of Na+
channels occur before the appearance of the septa109.
It should be noted that gradual detachment of the paranodal loops in the CNS of paranodal mutants is accompanied by the widening of the nodal gap and dispersion
of nodal Na+ channels. This indicates that, although the
septa are not required for the initial assembly of Na+
channels at the nodes, stabilized glial contacts (which
depend on septa) at the paranodes might be necessary to
maintain these clusters93,104,113. Interestingly, clustering of
Na+ channels in the optic nerve of Caspr-null mice, which
lack the paranodal septa, is associated with adjacent K+
channel clusters, raising the possibility that Caspr2 and
Tag1 compensate at these sites for the absence of Caspr
and contactin104.
In contrast to the clustering of Na+ channels at the
nodes, the formation of septa-containing axoglial junctions is essential for sequestering K+ channels at the juxtaparanodes86,87,94,95. These observations indicate that, once
formed, the axoglial septate junction functions as a fence
that restricts the movement of these channels and other
molecules from beneath the myelin sheath towards the
nodes. They also imply that a molecular sieve operating
at the paranodes during the formation of the nodes
would have to change its properties after the paranodal
loops have been secured to the axon by the septate junction. The generation of this fence might be mediated by
the attachment of the Caspr/contactin complex to the
axonal cytoskeleton88, binding to a glial ligand, and the
assembly of specific lipid microdomains. Although the
contribution of the lipid composition of the membrane
to the generation of axonal domains is yet to be investigated, it is of interest that contactin158, Caspr83 and Tag1
(REF. 159) are associated with rafts.
In addition to the paranodal junction, there might
also be a membrane barrier at the nodes. Although, the
Caspr2/K+ channel complex and Tag1 are aberrantly
located at the paranodal region in the absence of the paranodal junction, these proteins do not invade the nodes,
indicating the existence of an additional barrier at this
site86,87,94. A nodal barrier might be similar to the diffusion
barrier (or a membrane fence) that is found at the axon
initial segment, which could be regarded as the first node
in most myelinated axons160. At the axon initial segment,
this fence is formed by a high local concentration of
transmembrane proteins that are anchored to the actin
cytoskeleton and that serve as pickets, which can block the
diffusion of membrane proteins and phospholipids161.
Interestingly, an intact actin cytoskeleton in retinal
ganglion axons is also required for the clustering of
Na+ channels by a soluble factor that is secreted from
oligodendrocytes21.
Two other molecular mechanisms that might operate
in the formation of the nodes should be considered.
In the PNS, clustering of nodal Na+ channels during
development could also be mediated by contacting glial
processes that ‘drag’ Na+ channels and Ig-CAMs towards
their final position on the axolemma (FIG. 5b). This might
be mediated by binding of Na+ channels to the Schwann
cell microvilli, either directly through their β-subunits, or
indirectly through Nrcam and Nf186 (REF. 150). During
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development, the ERM-positive Schwann cell microvilli
make early contact with the nodes during their
formation54. These contact-sites (termed ‘caps’) contain
the phosphorylated adaptor EBP50 and face across
axonal ankyrin G53. Disruption of microvilli in mice
lacking Schwann cell dystroglycan resulted in a striking
reduction in clustering of nodal Na+ channels63. It
remains to be seen whether dystroglycan binds any of
the nodal proteins, thereby mediating this axoglial
interaction.
The microvilli also contain other candidate proteins,
including L1 (REF. 162) and neurofascin136, both of which
can bind Ig-CAMs present at the axolemma. Finally, it is
possible that the clustering of Na+ channels to the nodes is
mediated by downregulation of these channels from
beneath the internodes, and by the selective insertion of
newly synthesized or recycled molecules to the forming
nodal gap (FIG. 5d). Although it is less likely to operate during early development, a specific nodal delivery machinery is anticipated to exist, as indicated by the observations
that Na+ channel isoforms are replaced after the nodes
have been formed22,148, the presence of a high concentration of vesicles at the nodes163, and the close association of
Na+ channels with microtubules18. Similar mechanisms
might also operate in the formation of the juxtaparanodes in the CNS where K+ channels are first detected
during development117,145.
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The identification of a growing number of molecules
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provided an initial insight into the mechanisms that are
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Acknowledgements
We thank many colleagues for their input and discussion. We are
grateful to P. Shrager for his contribution of the computational model
presented in Fig. 3. The work carried out in the authors’ laboratory is
supported by the National Multiple Sclerosis Society, the United
States–Israel Science Foundation (BSF), the Minerva Foundation
and the Israel Science Foundation. E.P. is an Incumbent of the
Madeleine Haas Russell Career Development Chair.
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
ankyrin G | Caspr | contactin | Cx29 | dystroglycan | ezrin | moesin
| neurexins | neurofascin | Nrcam | phosphacan | protein 4.1B |
PSD-95 | radixin | Schwanomin | spectrin βIV | TAG-1 | tenascin-C
| tenascin-R | versican
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